Cellular adaptive immunity is mediated by T-lymphocytes, also known as T-cells, which upon recognition of a non-self or tumoral antigen can either destroy the target cell or orchestrate an immune response with other cells of the immune system.
Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T-cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T-cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T-cells or redirection of T-cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T-cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T-cells has been shown to be successful in treating melanoma.
Novel specificities in T-cells have been successfully generated through the genetic transfer of transgenic T-cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T cell cytotoxicity, however, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134), and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR modified T-cells. CARs have successfully allowed T-cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010).
While cytotoxic T-lymphocyte (CTL; also known as cytotoxic T-cells) and T helper cells play a central role in the cellular immune response, regulatory T-cells (Tregs), formerly known as suppressor T-cells, modulate or suppress immune responses, particularly to prevent autoimmunity and maintain tolerance to self-antigen. Because of their immune regulatory function, the presence of regulatory T-cells at a cancer or infection site may hinder the induction of an immune response against cancer or infectious pathogens (Aandahl E. M. et al. (2004); Cabrera R. et al. (2004); Viguier M. et al. (2004); Woo E. Y. et al (2001)). Therefore, in certain pathogenic situations such as chronic infectious diseases or cancer it may be desirable to suppress the activity of regulatory T-cells to allow a more potent immune response to occur. On another hand, vaccine strategies were developed based on the finding that vaccine efficacy could be improved by reducing the activity of regulatory T-cells, for instance by controlling the activity of the forkhead/winged helix transcription factor 3 (FoxP3). In particular, a peptide inhibitor of FOXP3, called P60, was found to improve vaccine efficacy in mice (Casares et al., 2010).
Dysfunction of FOXP3 is however associated with serious autoimmune disorders such as systemic lupus erythematosus or X-lined IPEX syndrome, such that systemic administration of inhibitors directed against, e.g, FoxP3, is currently not deemed a suitable option in immune therapy. The release of such inhibitors in the blood stream or even locally may lead to toxic effects by unleashing autoimmune reactions in organs not affected by the primary disease.
Accordingly, new therapeutic strategies are needed to facilitate an effective immune response while reducing possible toxic side effects in non-affected areas of the body. This need is addressed by the present invention by providing specific in-situ inhibition of regulatory T-cells as part of a CAR immunotherapy.